December 5, 2000 - VOL. 27, NO. 24

UT graduate students' innovation speeds biological research by up to 1,000 times

Three UT Austin graduate students have found a new use for an older cell sorting technology -- increasing the speed at which engineers and scientists can look for useful enzymes by up to 1,000 times. The discovery has landed the students in the prestigious international journal Nature Biotechnology and has major implications for the development of new enzymes for use in science and industry.

Working with millions of newly-formed enzymes, the student team, consisting of an engineer and two scientists, developed a method to make enzymes with desired traits flouresce, with the result that that they could be sorted out with a Florescence Activated Cell Sorting Machine. This decades-old machine originally was intended to sort cells in the human body and had never been used for studying enzyme variations before.

This machine can hone in on the desired enzymes far more rapidly than scientists can do so using microscopes. Patrick Daugherty, a graduate of the chemical engineering doctoral program, Mark Olsen, a doctoral candidate in chemistry and Daren Stephens, a masters candidate in molecular biology, previously had used the standard method of mutating and sorting new enzymes.

Enzymes act as biological catalysts, starting and accelerating chemical reactions in cells. Pharmaceutical companies, the plastics industry, environmental engineers and others work to develop new enzymes to perform a variety of tasks. Practical applications range from creating new beers to developing new cancer drugs.

Until now, the process of isolating desired enzymes required days or weeks, as scientists used microscopes manually to sort through millions of enzyme variants 1,000 cells at a time.

With the help of their supervising professors, Dr. George Georgiou and Dr. Brent Iverson, the students sought to automate the process using the Florescence Activated Cell Sorting Machine already available in their lab. Georgiou is a professor of chemical engineering and Iverson is an associate professor of chemistry and biochemistry.

The UT Austin researchers created a process using this machine that boosted their ability to screen for better enzymes by as much as 10 million enzymes per hour.

"If we can reprogram enzymes, then making complex molecules such as pharmaceuticals or precursors to polymers would be as easy as brewing beer. That is the long-term goal. But to get there, we have to make the enzymes do the right chemistry, and that's where we're working," said Iverson.

"And when we can find an accurate method of speeding this tedious process, then cancer cures and biodegradable plastics appear that much sooner in the future."

"Our process allows us to probe for the function of the mutant enzyme at the single cell level. There is no longer a need to grow large amounts of each of the cells producing different mutant enzymes separately," Georgiou said. "The larger the number of enzyme variants analyzed, the greater the probability that we will find an enzyme with the desired properties."

The researchers first employed a common process in enzyme science. They took an enzyme with a known function -- one of the many useful enzymes that cleave (or cut) bonds in other proteins. They then sprinkled mutations across the genes in the bacterial cells that produce the enzyme, resulting in mutated enzymes.

This produced a million enzyme variants, all different, so that the researchers still had to identify the enzymes doing the chemistry that they wanted.

The researchers accomplished this feat by putting the enzyme-producing bacteria -- harmless e. coli -- in a solvent with fluorescent molecules that were prevented from fluorescing by "quencher" molecules. When the cleaving protein was present in bacterial cells, it cut the fluorescent molecules from the quenchers, and they began to fluoresce.

The fluorescing molecules stuck to the cells that had produced the cleaving enzyme so that the researchers could identify the cells with that trait.

Instead of searching for telltale fluorescent cells approximately 1000 cells at a time with a microscope, the UT Austin scientists used the Florescence Activated Cell Sorting Machine to look at a million cells at a time.

"You take the sample of cells, and it sends them single file into a little chamber. They're hit by a light laser, and then each one is analyzed for the presence or absence of fluorescence," explained Iverson. "It takes the fluorescent cells and sticks them in a vial to collect and grow more cells. This happens ten million times an hour."

This method could run from 100 to 1000 times faster than previous technologies, and it here resulted in an enzyme that acted 60 times faster than the original enzyme after only one round of treatment," Iverson said.

The research was funded by the Welch Foundation, DARPA, the Department of Energy and the National Science Foundation.